Fused cyclooctyne compounds and their use in metal-free click reactions

ABSTRACT

The invention relates to fused cyclooctyne compounds, and to a method for their preparation. The invention also relates to a conjugate wherein a fused cyclooctyne compound according to the invention is conjugated to a label, and to the use of these conjugates in bioorthogonal labeling, imaging and/or modification, such as for example surface modification, of a target molecule. The invention further relates to a method for the modification of a target molecule, wherein a conjugate according to the invention is reacted with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/643,546, filed as a National Phase of International PatentApplication No. PCT/NL2011/050280, filed Apr. 26, 2011, published as WO2011/136645, which claims priority to European Application No.10161192.9 and U.S. Provisional Application No. 61/328,306, both filedApr. 27, 2010. The contents of these applications are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to fused cyclooctyne compounds and to a method fortheir preparation. The fused cyclooctyne compounds according to theinvention may be used in metal-free click reactions. Therefore, theinvention further relates to a method for the modification of a targetmolecule by reaction of a fused cyclooctyne conjugate with a targetmolecule comprising a 1,3-dipole or a 1,3-(hetero)diene. The inventionalso relates to the use of cyclooctyne conjugates in bioorthogonallabeling, imaging or modification of a target molecule.

BACKGROUND OF THE INVENTION

A revolutionary development in the rapidly expanding field of “chemicalbiology” is related to chemistry in living systems. Chemistry in livingsystems concerns chemical reactions that are mild in nature, yet sorapid and high-yielding that they work at about physiological pH, inwater, and in the vicinity of biomolecular functionalities. Suchreactions may be grouped under the term “bioorthogonal chemistry”. Inthe field of bioorthogonal chemistry there are two main challenges:first, the development of suitable chemistry, and second, theapplication thereof in living organisms (in vivo).

In the field of chemistry, an enormous toolbox of chemical reactions isavailable that may be applied to the construction of complex organicmolecules. However, the vast majority of such reactions can only beperformed under strictly anhydrous conditions, in other words, in thecomplete absence of water. Although still a good minority of chemicalreactions may be performed in, or in the presence of, water, most ofthese reactions can still only be applied in vitro because theinterference of other compounds present in the living organism with thechemicals involved can not be excluded. At present, only a handful ofchemical reactions is fully compatible with other functional groupspresent in the living organism.

An example of such a reaction is the cycloaddition of cyclic alkynes andazides, one of the reactions known as “click reactions”. This reactionhas become a versatile tool for bioorthogonal labeling and imaging ofbiomolecules (such as for example proteins, lipids, glycans and thelike), proteomics and materials science. In essence, two separatemolecular entities, one charged with an azide, and one charged with astrained cycloalkyne, will spontaneously combine into a single moleculeby a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC).The power of SPAAC for bioorthogonal labeling lies in the fact that anisolated cyclic alkyne or azide is fully inert to biologicalfunctionalities, such as for example amines, thiols, acids or carbonyls,but in combination undergo rapid and irreversible cycloaddition leadingto a stable triazole conjugate. For example, azido-modified proteins,obtained by expression in auxotrophic bacteria, genetic engineering orchemical conversion, can be cleanly labeled with biotin, fluorophores,PEG-chains or other functionalities upon simply stirring theazido-protein with a cyclooctyne conjugate. Moreover, the small size ofazide has proven highly useful for application of SPAAC in the imagingof specific biomolecules by means of the chemical reporter strategy.

Apart from azides, cyclooctynes also show high reactivity with otherdipoles, such as nitrones and nitrile oxides. For example, thestrain-promoted alkyne-nitrone cycloaddition (SPANC) was applied for theN-terminal modification of proteins.

SPAAC and SPANC cycloaddition reactions (Scheme 1) proceedspontaneously, hence in the absence of a (metal) catalyst, and these anda select number of additional cycloadditions are also referred to as“metal-free click reactions”.

Several cyclic alkynes and their application in bioorthogonal labelingare described to in the prior art. US 2009/0068738, incorporated byreference, relates to modified cycloalkyne compounds and their use inmodifying biomolecules via a cycloaddition reaction that may be carriedout under physiological conditions. The cycloaddition involves reactinga modified cycloalkyne, such as for example difluorinated cyclooctynecompounds DIFO, DIFO2 and DIFO3, with an azide moiety on a targetbiomolecule, generating a covalently modified biomolecule. It wasobserved that fluoride substitution has an accelerating effect on thecycloaddition with azide. For example DIFO3 displays a significantlyimproved reaction rate constant of up to k=76×10⁻³ M⁻¹ s⁻¹, versus amaximum of 2.4×10⁻³ M⁻¹ s⁻¹ for non-fluorinated systems.

Cyclooctynes wherein the cyclooctyne is fused to aryl groups(benzannulated systems) are disclosed in WO 2009/067663, incorporated byreference, and the reaction kinetics of these dibenzocyclooctynecompounds DIBO in the cycloaddition with azides are further improved(k=0.12 M⁻¹ s⁻¹).

Azadibenzocyclooctyne DIBAC was developed by van Delft et al. (Chem.Commun. 2010, 46, 97-99), incorporated by reference, and shows furtherimproved reaction kinetics in the cycloaddition with azides (k=0.31 M⁻¹s⁻¹).

Recently another benzannulated system, biarylazacyclooctynone BARAC, wasreported by Bertozzi et al. (J. Am. Chem. Soc. 2010, 132, 3688-3690),incorporated by reference. By placing the amide functionality in thering, the reaction kinetics of the cycloaddition of BARAC with azideswas improved significantly (k=0.96 M⁻¹ s⁻¹).

DIBO and DIBAC were also found to undergo rapid cycloaddition withnitrones as described by Pezacki (Chem. Common. 2010, 46, 931-933) andby van Delft (Angew. Chem. Int. Ed. 2010, 49, 3065-3068), bothincorporated by reference, with reaction rate constants up to 300 timeshigher than with azides.

However, the cyclooctyne probes for bioorthogonal labeling known in theprior art suffer from several disadvantages. First of all, widespreadapplication is hampered by the fact that only DIBAC is commerciallyavailable. Synthetic preparation requires advanced chemical expertise.In addition, synthesis of the currently available probes is lengthy(eight chemical steps for DIFO2, ten steps for DIFO3, nine steps forDIBAC), and/or low-yielding (10% overall for DIBO). Thirdly, thepresence of the two benzannulated aryl moieties in DIBO and DIBACinflicts both serious steric repulsion as well as lipophilic character.The lipophilic character of DIBO and DIBAC may lead to a specificprotein binding by van der Waals interactions, which is undesirable.

Hence, there exists a clear demand for novel, readily accessible andreactive bioorthogonal probes for use in metal-free click reactions,such as 1,3-dipolar cycloaddition with azides, nitrones and other1,3-dipoles.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a compound of theFormula (Ia), (Ib) or (Ic),

-   -   wherein:    -   m is 0 or 1;    -   n is 0 to 8;    -   p is 0 or 1;    -   Z is N or C(R³), wherein R³ is selected from the group        consisting of [(L)_(p)-Q], hydrogen, halogen, C₁-C₂₄ alkyl        groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl        groups and C₇-C₂₄ (hetero)arylalkyl groups, the alkyl groups        optionally being interrupted by one of more hetero-atoms        selected from the group consisting of O, N and S, wherein the        alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and        (hetero)arylalkyl groups are independently optionally        substituted with one or more substituents independently selected        from the group consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂ alkenyl        groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₁-C₁₂        alkoxy groups, C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy        groups, C₃-C₁₂ cycloalkyloxy groups, halogens, amino groups, oxo        groups and silyl groups, wherein the alkyl groups, alkenyl        groups, alkynyl groups, cycloalkyl groups, alkoxy groups,        alkenyloxy groups, alkynyloxy groups and cycloalkyloxy groups        are optionally substituted, the alkyl groups, the alkoxy groups,        the cycloalkyl groups and the cycloalkoxy groups being        optionally interrupted by one of more hetero-atoms selected from        the group consisting of O, N and S, wherein the silyl groups are        represented by the formula (R⁴)₃Si—, wherein R⁴ is independently        selected from the group consisting of C₁-C₁₂ alkyl groups,        C₂-C₁₂ alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl        groups, C₁-C₁₂ alkoxy groups, C₂-C₁₂ alkenyloxy groups, C₂-C₁₂        alkynyloxy groups and C₃-C₁₂ cycloalkyloxy groups, wherein the        alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups,        alkoxy groups, alkenyloxy groups, alkynyloxy groups and        cycloalkyloxy groups are optionally substituted, the alkyl        groups, the alkoxy groups, the cycloalkyl groups and the        cycloalkoxy groups being optionally interrupted by One of more        hetero-atoms selected from the group consisting of O, N and S;    -   Y is O, C(O) or C(R⁵)₂, wherein R⁵ is independently selected        from the group consisting of hydrogen, halogen, C₁-C₂₄ alkyl        groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl        groups, and C₇-C₂₄ (hetero)arylalkyl groups, the alkyl groups        optionally being interrupted by one of more hetero-atoms        selected from the group consisting of O, N and S, wherein the        alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and        (hetero)arylalkyl groups are optionally substituted;    -   optionally, when m=1, Y and Z together with the bond connecting        Y and Z, form a cyclic alkyl group or a (hetero)aryl group,        wherein the cyclic alkyl group and the (hetero)aryl group are        optionally substituted, provided that when Y and Z form a        (hetero)aryl group, then the group -(L)_(p)-Q is a substituent        on the (hetero)aryl group;    -   L is a linking group selected from linear or branched C₁-C₂₄        alkylene groups, C₂-C₂₄ alkenylene groups, C₂-C₂₄ alkynylene        groups, C₃-C₂₄ cycloalkylene groups, C₅-C₂₄ cycloalkenylene        groups, C₈-C₂₄ cycloalkynylene groups, C₇-C₂₄        alkyl(hetero)arylene groups, C₇-C₂₄ (hetero)arylalkylene groups.        C₈-C₂₄ (hetero)arylalkenylene groups, C₉-C₂₄        (hetero)arylalkynylene groups, the alkylene groups, alkenylene        groups, alkynylene groups, cycloalkylene groups, cycloalkenylene        groups, cycloalkynylene groups, alkyl(hetero)arylene groups,        (hetero)arylalkylene groups, (hetero)arylalkenylene groups and        (hetero)arylalkynylene groups optionally being substituted with        one or more substituents independently selected from the group        consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂ alkenyl groups, C₂-C₁₂        alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₅-C₁₂ cycloalkenyl        groups, C₈-C₁₂ cycloalkynyl groups, C₁-C₁₂ alkoxy groups, C₂-C₁₂        alkenyloxy groups, C₂-C₁₂ alkynyloxy groups, C₃-C₁₂        cycloalkyloxy groups, halogens, amino groups, oxo and silyl        groups, wherein the silyl groups can be represented by the        formula (R⁴)₃Si—, wherein R⁴ is defined as above;    -   Q is a functional group selected from the group consisting of        hydrogen, halogen, R⁶, —CH═C(R⁶)₂, —C≡CR⁶,        —[C(R⁶)₂C(R⁶)₂O]_(q)—R⁶, wherein q is in the range of 1 to 200,        —CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)_(3,) —C(X)N(R⁶)₂,        —C(R⁶)₂XR⁶, —C(X)R⁶, —C(X)XR⁶, —S(O)R⁶, —S(O)₂R⁶, —S(O)OR⁶,        —S(O)₂OR⁶, —S(O)N(R⁶)₂, —S(O)₂N(R⁶)₂, —OS(O)R⁶, —OS(O)₂R⁶,        —OS(O)OR⁶, —OS(O)₂OR⁶, —P(O)(R⁶)(OR⁶), —P(O)(OR⁶)₂,        —OP(O)(OR⁶)₂, —Si(R⁶)₃, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂,        —N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, wherein X is        oxygen or sulphur and wherein R⁶ is independently selected from        the group consisting of hydrogen, halogen. C₁-C₂₄ alkyl groups,        C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl groups and        C₇-C₂₄ (hetero)arylalkyl groups;    -   R¹ is independently selected from the group consisting of        hydrogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups,        C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl        groups; and    -   R² is independently selected from the group consisting of        halogen, —OR⁶, —NO₂, —CN, —S(O)₂R⁶, C₁-C₁₂ alkyl groups, C₁-C₁₂        aryl groups, C₁-C₁₂ alkylaryl groups and C₁-C₁₂ arylalkyl        groups, wherein R⁶ is as defined above, and wherein the alkyl        groups, aryl groups, alkylaryl groups and arylalkyl groups are        optionally substituted.

The present invention further relates to a conjugate, such as acyclooctyne conjugate, wherein a compound of the Formula (Ia), (Ib)and/or (Ic) is conjugated to a label via a functional group Q.

Another aspect of the present invention is to provide a method forpreparing a compound of the general Formula (Ia), (Ib) or (Ic), themethod comprising the steps of:

-   -   (a) Introduction of a fused 3- or 4-membered ring to a        cyclooctadiene of the Formula (VIIa), (VIIb) or (VIIc):

-   -   -   wherein:        -   n=0 to 8;        -   R¹ is independently selected from the group consisting of            hydrogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups,            C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl            groups; and        -   R² is independently selected from the group consisting of            halogen, —OR⁶, —NO₂, —CN, —S(O)₂R⁶, C₁-C₁₂ alkyl groups,            C₁-C₁₂ aryl groups, C₁-C₁₂ alkylaryl groups and C₁-C₁₂            arylalkyl groups, wherein the alkyl groups, aryl groups,            alkylaryl groups and arylalkyl groups are optionally            substituted, and wherein R⁶ is independently selected from            the group consisting of hydrogen, halogen, C₁-C₂₄ alkyl            groups, C₆-C₂₄ (hetero)aryl groups. C₇-C₂₄ alkyl(hetero)aryl            groups and C₇-C₂₄ (hetero)arylalkyl groups.

    -   to form a bicyclic cyclooctene compound,

    -   (b) Bromination of the obtained bicyclic cyclooctene compound to        form a bicyclic cyclooctane compound, and

    -   (c) Dehydrobromination of the obtained bicyclic cyclooctane        compound.

The present invention also relates to a method for the modification of atarget molecule, wherein a conjugate according to the invention isreacted with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene.

Yet another aspect of the present invention is the use of a conjugateaccording to the invention for bioorthogonal labeling, imaging ormodification, such as for example surface modification, of a targetmolecule.

Finally, the invention relates to a composition comprising a conjugateaccording to the invention, further comprising a pharmaceuticallyacceptable carrier.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the SDS-PAGE analyses of the SPAAC reaction ofazide-labeled capsid protein with a cyclooctyne conjugated to AlexaFluor 555 (left) and of the blank reaction (right).

FIG. 2 depicts the ESI-TOF mass spectrum of capsid proteinfunctionalized with a cyclooctyne conjugated to Alexa Fluor 555.

FIG. 3 depicts the FPLC analysis of capsin protein functionalized with acyclooctyne conjugated to Alexa Fluor 555.

FIG. 4 depicts the cell surface fluorescence on intact MV3 cells aftermetabolic incorporation of Ac₄ManNAz, labeling with DIBO- or BCN-biotin,and detection with AlexaFluor488-conjugated streptavidin as determinedwith flow cytometry.

FIGS. 5A and 5B depict the fluorescence intensities and cell viabilityof MV3 cells after metabolic incorporation of Ac₄ManNAz, labeling withDIBO- or BCN-biotin, and detection with AlexaFluor488-conjugatedstreptavidin.

FIG. 6 depicts representative confocal images of labeled cells,previously cultured in absence or presence of Ac₄ManNAz (50 μM),labeling with DIBO- or BCN-biotin, and detection withAlexaFluor488-conjugated streptavidin.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The verb “to comprise” as is used in this description and in the claimsand its conjugations is used in its non-limiting sense to mean thatitems following the word are included, but items not specificallymentioned are not excluded.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there is one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one”.

The compounds disclosed in this description and in the claims may bedescribed as fused cyclooctyne compounds, i.e. cyclooctyne compoundswherein a second ring structure is fused to the cyclooctyne moiety. Thetriple bond of the cyclooctyne moiety in a fused cyclooctyne compoundmay be located on either one of the three possible locations, i.e. onthe 2, 3 or 4 position of the cyclooctyne moiety (numbering according to“IUPAC Nomenclature of Organic Chemistry”, Rule A31.2). The descriptionof any fused cyclooctyne compound in this description and in the claimsis meant to include all three individual regioisomers of the cyclooctynemoiety.

The compounds disclosed in this description and in the claims maycomprise one or more asymmetric centres, and different diastereomersand/or enantiomers may exist of the compounds. The description of anycompound in this description and in the claims is meant to include alldiastereomers, and mixtures thereof, unless stated otherwise. Inaddition, the description of any compound in this description and in theclaims is meant to include both the individual enantiomers, as well asany mixture, racemic or otherwise, of the enantiomers, unless statedotherwise. When the structure of a compound is depicted as a specificenantiomer, it is to be understood that the invention of the presentapplication is not limited to that specific enantiomer.

The compounds may occur in different tautomeric forms. The compoundsaccording to the invention are meant to include all tautomeric forms,unless stated otherwise.

The compounds disclosed in this description and in the claims mayfurther exist as exo and endo regioisomers. Unless stated otherwise, thedescription of any compound in the description and in the claims ismeant to include both the individual exo and the individual endoregioisomer of a compound, as well as mixtures thereof.

Furthermore, the compounds disclosed in this description and in theclaims may exist as cis and trans isomers. Unless stated otherwise, thedescription of any compound in the description and in the claims ismeant to include both the individual cis and the individual trans isomerof a compound, as well as mixtures thereof. As an example, when thestructure of a compound is depicted as a cis isomer, it is to beunderstood that the corresponding trans isomer or mixtures of the cisand trans isomer are not excluded from the invention of the presentapplication.

Unsubstituted alkyl groups have the general formula C_(n)H_(2n+1) andmay be linear or branched. Unsubstituted alkyl groups may also contain acyclic moiety, and thus have the concomitant general formulaC_(n)H_(2n−1). Optionally, the alkyl groups are substituted by one ormore substituents further specified in this document. Examples ofsuitable alkyl groups include, but are not limited to, methyl, ethyl,propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl and the like.

Unsubstituted alkenyl groups have the general formula C_(n)H_(2n−1), andmay be linear or branched. Examples of suitable alkenyl groups include,but are not limited to, ethenyl, propenyl, isopropenyl, butenyl,pentenyl, decenyl, octadecenyl, and eicosenyl and the like.Unsubstituted alkenyl groups may also contain a cyclic moiety, and thushave the concomitant general formula C_(n)H_(2n−3).

Unsubstituted alkenes have the general formula C_(n)H_(2n) whereasunsubstituted alkynes have the general formula C_(n)H_(2n−2).

Aryl groups comprise at least six carbon atoms and may includemonocyclic, bicyclic and polycyclic structures. Optionally, the arylgroups may be substituted by one or more substituents further specifiedin this document. Examples of aryl groups include groups such as forexample phenyl, naphthyl, anthracyl and the like.

Arylalkyl groups and alkylaryl groups comprise at least seven carbonatoms and may include monocyclic and bicyclic structures. Optionally,the aryl groups may be substituted by one or more substituents furtherspecified in this document. An arylalkyl group is for example benzyl andthe like. An alkylaryl group is for example 4-t-butylphenyl and thelike.

Where an aryl group is denoted as a (hetero)aryl group, the notation ismeant to include an aryl group and a heteroaryl group. Similarly, analkyl(hetero)aryl group is meant to include an alkylaryl group and aalkylheteroaryl group, and (hetero)arylalkyl is meant to include anarylalkyl group and a heteroarylalkyl group.

A heteroaryl group comprises one to four heteroatoms selected from thegroup consisting of oxygen, nitrogen and sulphur.

Fused Cyclooctyne Compounds

Based on the fact that the reactivity of cycloalkynes increases withdecreasing ring size, conjugates of cycloalkynes with less than eightcarbon atoms would be of high interest for application in bioorthogonalchemistry. Unfortunately, to date cycloheptyne and smaller rings cannotbe isolated in pure form, due to increasing strain energy as a result ofdeviation from the ideal 180° C.—C≡C angle.

An alternative way to increase strain energy is by benzannulation ofcyclooctyne to aryl groups, the strategy followed in WO 2009/067663 forDIBO. The strain energy may then be further enhanced by introduction ofanother sp²-type atom in the ring, the approach followed by van Delftfor DIBAC and by Bertozzi for BARAC. However, as was mentioned above,the presence of two aryl moieties in cyclooctyne not only gives rise toserious steric repulsion, but also increases the lipophilic character ofthe cyclooctyne compounds, which is undesired.

The present inventors found that a very efficient way to induceadditional ring strain involves fusion of a cyclooctyne to a 3- or4-membered ring, leading to fused cyclooctyne compounds, more inparticular to bicyclo[6.1.0]nonyne and bicyclo[6.2.0]decyne systems,respectively. These bicyclic systems are surprisingly well suited asbioorthogonal probes, since they combine relative stability with a highreactivity in (3+2) cycloadditions with 1,3-dipoles and in (hetero)Diels-Alder reactions with 1,3-(hetero)dienes. Furthermore, the fused 3-or 4-membered ring structure, apart from inflicting ring strain, is alsoperfectly suitable for the positioning of a handle for the conjugationto functional groups and/or labels and can be convenientlyfunctionalized for applications such as bioorthogonal labeling, imagingand/or modification, such as for example surface modification, of targetmolecules.

In a first aspect, the present invention therefore relates to compoundsof the general Formula (Ia), (Ib) or (Ic),

-   -   wherein:    -   m is 0 or 1;    -   n is 0 to 8;    -   p is 0 or 1;    -   Z is N or C(R³), wherein R³ is selected from the group        consisting of [(L)_(p)-Q], hydrogen, halogen. C₁-C₂₄ alkyl        groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl        groups and C₇-C₂₄ (hetero)arylalkyl groups, the alkyl groups        optionally being interrupted by one of more hetero-atoms        selected from the group consisting of O, N and S, wherein the        alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and        (hetero)arylalkyl groups are independently optionally        substituted with one or more substituents independently selected        from the group consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂ alkenyl        groups, C₄-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₁-C₁₂        alkoxy groups, C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy        groups, C₃-C₁₂ cycloalkyloxy groups, halogens, amino groups, oxo        groups and silyl groups, wherein the alkyl groups, alkenyl        groups, alkynyl groups, cycloalkyl groups, alkoxy groups,        alkenyloxy groups, alkynyloxy groups and cycloalkyloxy groups        are optionally substituted, the alkyl groups, the alkoxy groups,        the cycloalkyl groups and the cycloalkoxy groups being        optionally interrupted by one of more hetero-atoms selected from        the group consisting of O, N and S, wherein the silyl groups are        represented by the formula (R⁴)₃Si—, wherein R⁴ is independently        selected from the group consisting of C₁-C₁₂ alkyl groups,        C₂-C₁₂ alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl        groups, C₁-C₁₂ alkoxy groups, C₂-C₁₂ alkenyloxy groups, C₂-C₁₂        alkynyloxy groups and C₃-C₁₂ cycloalkyloxy groups, wherein the        alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups,        alkoxy groups, alkenyloxy groups, alkynyloxy groups and        cycloalkyloxy groups are optionally substituted, the alkyl        groups, the alkoxy groups, the cycloalkyl groups and the        cycloalkoxy groups being optionally interrupted by one of more        hetero-atoms selected from the group consisting of O, N and S;    -   Y is O, C(O) or C(R⁵)₂, wherein R⁵ is independently selected        from the group consisting of hydrogen, halogen, C₁-C₂₄ alkyl        groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl        groups, and C₇-C₂₄ (hetero)arylalkyl groups, the alkyl groups        optionally being interrupted by one of more hetero-atoms        selected from the group consisting of O, N and S, wherein the        alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and        (hetero)arylalkyl groups are optionally substituted;    -   optionally, when m=1, Y and Z together with the bond connecting        Y and Z, form a cyclic alkyl group or a (hetero)aryl group,        wherein the cyclic alkyl group and the (hetero)aryl group are        optionally substituted, provided that when Y and Z form a        (hetero)aryl group, then the group -(L)_(p)-Q is a substituent        on the (hetero)aryl group;    -   L is a linking group selected from linear or branched C₁-C₂₄        alkylene groups, C₂-C₂₄ alkenylene groups, C₂-C₂₄ alkynylene        groups, C₃-C₂₄ cycloalkylene groups, C₅-C₂₄ cycloalkenylene        groups, C₈-C₂₄ cycloalkynylene groups, C₇-C₂₄        alkyl(hetero)arylene groups, C₇-C₂₄ (hetero)arylalkylene groups,        C₈-C₂₄ (hetero)arylalkenylene groups, C₉-C₂₄        (hetero)arylalkynylene groups, the alkylene groups, alkenylene        groups, alkynylene groups, cycloalkylene groups, cycloalkenylene        groups, cycloalkynylene groups, alkyl(hetero)arylene groups,        (hetero)arylalkylene groups, (hetero)arylalkenylene groups and        (hetero)arylalkynylene groups optionally being substituted with        one or more substituents independently selected from the group        consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂ alkenyl groups, C₂-C₁₂        alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₅-C₁₂ cycloalkenyl        groups, C₈-C₁₂ cycloalkynyl groups, C₁-C₁₂ alkoxy groups, C₂-C₁₂        alkenyloxy groups, C₂-C₁₂ alkynyloxy groups, C₃-C₁₂        cycloalkyloxy groups, halogens, amino groups, oxo and silyl        groups, wherein the silyl groups can be represented by the        formula (R⁴)₃Si—, wherein R⁴ is defined as above;    -   Q is a functional group selected from the group consisting of        hydrogen, halogen, R⁶, —CH═C(R⁶)₂, —C═CR⁶,        —[C(R⁶)₂C(R⁶)₂O]_(q)—R⁶, wherein q is in the range of 1 to 200,        —CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)₃, —C(X)N(R⁶)₂,        —C(R⁶)₂XR⁶, —C(X)R⁶, —C(X)XR⁶, —S(O)R⁶, —S(O)₂R⁶, —S(O)OR⁶,        —S(O)₂OR⁶, —S(O)N(R⁶)₂, —S(O)₂N(R⁶)_(2,) —OS(O)R⁶, —OS(O)₂R⁶,        —OS(O)OR⁶, —OS(O)₂OR⁶, —P(O)(R⁶)(OR⁶), —P(O)(OR⁶)₂,        —OP(O)(OR⁶)₂, —Si(R⁶)₃, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂,        —N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, wherein X is        oxygen or sulphur and wherein R⁶ is independently selected from        the group consisting of hydrogen, halogen, C₁-C₂₄ alkyl groups,        C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl groups and        C₇-C₂₄ (hetero)arylalkyl groups;    -   R¹ is independently selected from the group consisting of        hydrogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups,        C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl        groups; and    -   R² is independently selected from the group consisting of        halogen, —OR⁶, —NO₂, —CN, —S(O)₂R⁶, C₁-C₁₂ alkyl groups, C₁-C₁₂        aryl groups, C₁-C₁₂ alkylaryl groups and C₁-C₁₂ arylalkyl        groups, wherein R⁶ is as defined above, and wherein the alkyl        groups, aryl groups, alkylaryl groups and arylalkyl groups are        optionally substituted.

An advantage of the fused cyclooctyne compounds according to the presentinvention is that they are easily synthesized. A further advantage isthat they are amenable to simple and straightforward modification of thevarious parts of the molecule.

The fused cyclooctyne compound of Formula (Ia) is preferred. In thiscompound, the triple bond is located on the 4-position of thecyclooctyne moiety, i.e. opposite to the fused 3- or 4-membered ring.This may result in a decreased number of potential regioisomers possiblefor (Ia), depending on the nature of the substituents on the cyclooctynecompound.

In the compounds of Formula (Ia), (Ib) and (Ic), the -[(L)_(p)-Q]substituent on Z may be positioned on the exo or on the endo positionrespective to the cyclooctyne ring. The two R¹-substituents may be in acis or in a trans position relative to each other.

Preferably, Q is selected from the group consisting of —CN, —N₃, —NCX,—XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)₃, —C(X)N(R⁶)₂, —C(R⁶)₂XR⁶, —C(X)R⁶,—C(X)XR⁶, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂, —N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶and —N(R⁶)C(X)N(R⁶)₂, wherein X and R⁶ are as defined above. Morepreferably, X is oxygen. Most preferably, Q is selected from the groupconsisting of —OR⁶, —N(R⁶)₂, —⁺N(R⁶)₃, —C(O)N(R⁶)₂, —C(O)OR⁶, —OC(O)R⁶,—OC(O)OR⁶, —OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶, —N(R⁶)C(O)OR⁶ and—N(R⁶)C(O)N(R⁶)₂. Furthermore, the functional group Q may optionally bemasked or protected. The R⁶ groups may be selected independently fromeach other, which means that the two R⁶ groups present in, for example,a —N(R⁶)₂ substituent may be different from each other.

In one embodiment, p is 0, i.e. Q is bonded directly to Z. In anotherembodiment, p is 1. In yet another embodiment, p is 1 and L is CH₂.

In a preferred embodiment, R₁ is hydrogen.

In another preferred embodiment, n is 0. In yet another preferredembodiment, R² is an electron-withdrawing group, i.e. a group with apositive value for the Hammett substituent constant σ. Suitableelectron-withdrawing groups are known to a person skilled in the art,and include for example halogen (in particular F), —OR⁶, —NO₂, —CN,—S(O)₂R⁶, substituted C₁-C₁₂ alkyl groups, substituted C₁-C₁₂ arylgroups or substituted C₁-C₁₂ alkylaryl groups, wherein the substituentsare electron-withdrawing groups. Preferably, the substituted alkylgroups, aryl groups and alkylaryl groups are fluorinated C₁-C₁₂ alkylgroups (such as for example —CF₃), fluorinated C₁-C₁₂ aryl groups (suchas for example —C₆F₅) or fluorinated C₁-C₁₂ alkylaryl groups (such asfor example -[3,5-(CF₃)₂(C₆H₃)]).

Compounds with m=0 and Z═CR³

A specific class of compounds the present invention relates to is theclass of compounds according to Formula (Ia), (Ib) or (Ic) wherein thecyclooctyne moiety is fused to a three-numbered ring structure.Therefore, in a preferred embodiment of the present invention m, asdefined above, is 0.

In a further preferred embodiment, Z is C(R³), wherein R³ is as definedabove. The present invention therefore also relates to a compoundaccording to Formula (Ia), (Ib) or (Ic), wherein the compound is of theFormula (IIa), (IIb) or (IIc):

wherein n, p, R¹, R², R³, L and Q are as defined above for compounds ofthe Formula (Ia), (Ib) and (Ic).

As was already mentioned above, the -[(L)_(p)-Q] substituent in thecompounds of Formula (IIa), (IIb) and (IIc), may be positioned exo orendo with respect to the cyclooctyne ring, and the two R¹ substituentsmay be positioned in a cis or in a trans position relative to eachother. In a preferred embodiment, the R¹ substituents are in a cisposition.

In one embodiment, p is 0, i.e. Q is bonded directly to the cyclopropylring. In another embodiment, p is 1. In yet another embodiment, p is 1and F is CH₂.

Also in compounds of the Formula (IIa), (IIb) or (IIc), both when p is 0and when p is 1, Q is preferably selected from the group consisting of—CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)₂, —C(R⁶)₂XR⁶, —C(X)R⁶,—C(X)XR⁶, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂, —N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶and —N(R⁶)C(X)N(R⁶)₂, wherein X and R⁶ are as defined above. Morepreferably, X is oxygen. Most preferably, Q is selected from the groupconsisting of —OR⁶, —N(R⁶)₂, —⁺N(R⁶)₃, —C(O)N(R⁶)₂, —C(O)OR⁶, —OC(O)R⁶,—OC(O)OR⁶, —OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶, —N(R⁶)C(O)OR⁶ and—N(R⁶)C(O)N(R⁶)₂. Furthermore, the functional group Q may optionally bemasked or protected. The R⁶ groups may be selected independently fromeach other, which means that the two R⁶ groups present in, for example,a —N(R⁶)₂ substituent may be different from each other. In a preferredembodiment, Q is —OR⁶, preferably —OH. In another preferred embodiment,Q is —C(O)OR⁶.

In another preferred embodiment, R³ is hydrogen or [(L)_(p)-Q].

In a preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, n is 0. In yet another preferredembodiment, R² is an electron-withdrawing group, i.e. a group with apositive value for the Hammett substituent constant σ. Suitableelectron-withdrawing groups are known to a person skilled in the art,and include for example halogen (in particular F), —OR⁶, —NO₂, —CN,—S(O)₂R⁶, substituted C₁-C₁₂ alkyl groups, substituted C₁-C₁₂ arylgroups or substituted C₁-C₁₂ alkylaryl groups, wherein the substituentsare electron-withdrawing groups. Preferably, the substituted alkylgroups, aryl groups and alkylaryl groups are fluorinated C₁-C₁₂ alkylgroups (such as for example —CF₃), fluorinated C₁-C₁₂ aryl groups (suchas for example —C₆F₅) or fluorinated C₁-C₁₂ alkylaryl groups (such asfor example -[3,5-(CF₃)₂(C₆H₃)]).

Of the compounds of Formula (IIa), (IIb) and (IIc), compounds of theFormula (IIa) are preferred since they may possess an axis or a plane ofsymmetry, thus reducing the number of potential regioisomers that may beformed.

An embodiment wherein p is 1, L is CH₂, Q is —OH, R¹ is hydrogen, R³ ishydrogen or [(L)_(p)-Q] and n is 0 is particularly preferred.

Compounds with m=0 and Z═N

The present invention also relates to compounds of the general Formula(Ia), (Ib) or (Ic), wherein m is 0 and Z is N. These compounds have thegeneral Formula (IIIa), (IIIb) or (IIIc):

wherein n, p, R¹, R², L and Q are as defined above for compounds of theFormula (Ia), (Ib) and (Ic).

The two R¹ substituents may be positioned in a cis or in a transposition relative to each other. In a preferred embodiment, the R¹substituents are in a cis position.

In one embodiment, p is 0, i.e. Q is bonded directly to N. In anotherembodiment, p is 1. In yet another embodiment, p is 1 and L is CH₂.

Also in compounds of the Formula (IIIa), (IIIb) or (IIIc), both when pis 0 and when p is 1, Q is preferably selected from the group consistingof —CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)_(3,) —C(R⁶)₂XR⁶,—C(X)R⁶, —C(X)XR⁶, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂, —N(R⁶)C(X)R⁶,—N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, wherein X and R⁶ are as definedabove. More preferably, X is oxygen. Most preferably, Q is selected fromthe group consisting of —OR⁶, —N(R⁶)₂, —⁺N(R⁶)₃, —C(O)N(R⁶)₂, —C(O)OR⁶,—OC(O)R⁶, —OC(O)OR⁶, —OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶, —N(R⁶)C(O)OR⁶ and—N(R⁶)C(O)N(R⁶)₂. Furthermore, the functional group Q may optionally bemasked or protected. The R⁶ groups may be selected independently fromeach other, which means that the two R⁶ groups present in, for example,a —N(R⁶)₂ substituent may be different from each other.

In a preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, n is 0. In yet another preferredembodiment, R² is an electron-withdrawing group, i.e. a group with apositive value for the Hammett substituent constant σ. Suitableelectron-withdrawing groups are known to a person skilled in the art,and include for example halogen (in particular F), —OR⁶, —NO₂, —CN,—S(O)₂R⁶, substituted C₁-C₁₂ alkyl groups, substituted C₁-C₁₂ arylgroups or substituted C₁-C₁₂ alkylaryl groups, wherein the substituentsare electron-withdrawing groups. Preferably, the substituted alkylgroups, aryl groups and alkylaryl groups are fluorinated C₁-C₁₂ alkylgroups (such as for example —CF₃), fluorinated C₁-C₁₂ aryl groups (suchas for example —C₆F₅) or fluorinated C₁-C₁₂ alkylaryl groups (such asfor example -[3,5-(CF₃)₂(C₆H₃)]).

Of the compounds of Formula (IIIa), (IIIb) and (IIIc), compounds of theFormula (IIIa) are preferred since they possess an element of symmetry,thus reducing the number of potential regioisomers that may be formed.

Compounds with m=1 and Z═C(R³)

Furthermore, the present invention relates to compounds of the generalFormula (Ia), (Ib) or (Ic) wherein m is 1 and Z is C(R³), and thesecompounds have the general Formula (IVa), (IVb) or (IVc):

wherein Y is O, C(O) or C(R⁵)₂, and n, p, L, Q, R¹, R², R³ and R⁵ are asdefined above.

Also in the compounds of Formula (IVa), (IVb) and (IVc), the-[(L)_(p)-Q] substituent may be positioned exo or endo with respect tothe cyclooctyne ring, and the two R¹ substituents may be positioned in acis or in a trans position relative to each other.

In the compounds of Formula (IVa), (IVb) and (IVc) as depicted above,the Z-group, i.e. the C-atom bonded to R¹ and to the -[(L)_(p)-Q]substituent, is located at the 9-position of the fused cyclooctynecompound. However, the regioisomers of (IVb) and (IVc) wherein thisZ-group is positioned on the 10-position of the fused bicyclodecynecompound are also included, whereby the -[(L)_(p)-Q] substituent may bepositioned on the exo or the endo position.

In one embodiment, p is 0, i.e. Q is bonded directly to the cyclobutylring. In another embodiment, p is 1. In yet another embodiment, p is 1and L is CH₂.

Also in compounds of the Formula (IVa), (IVb) or (IVc), both when p is 0and when p is 1, Q is preferably selected from the group consisting of—CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)_(3,) —C(X)N(R⁶)₂,—C(R⁶)₂XR⁶, —C(X)R⁶, —C(X)XR⁶, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂,—N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, wherein X and R⁶ areas defined above. More preferably, X is oxygen. Most preferably, Q isselected from the group consisting of —OR⁶, —N(R⁶)₂, —⁺N(R⁶)₃,—C(O)N(R⁶)₂, —C(O)OR⁶, —OC(O)R⁶, —OC(O)OR⁶, —OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶,—N(R⁶)C(O)OR⁶ and —N(R⁶)C(O)N(R⁶)₂. Furthermore, the functional group Qmay optionally be masked or protected. The R⁶ groups may be selectedindependently from each other, which means that the two R⁶ groupspresent in, for example, a —N(R⁶)₂ substituent may be different fromeach other.

In a preferred embodiment, Y is O. In another preferred embodiment, Y isC(O). Both when Y is O and when Y is C(O), it is preferred that R¹ ishydrogen.

In another preferred embodiment, n is 0. In yet another preferredembodiment, R² is an electron-withdrawing group, i.e. a group with apositive value for the Hammett substituent constant σ. Suitableelectron-withdrawing groups are known to a person skilled in the art,and include for example halogen (in particular F), —OR⁶, —NO₂, —CN,—S(O)₂R⁶, substituted C₁-C₁₂ alkyl groups, substituted C₁-C₁₂ arylgroups or substituted C₁-C₁₂ alkylaryl groups, wherein the substituentsare electron-withdrawing groups. Preferably, the substituted alkylgroups, aryl groups and alkylaryl groups are fluorinated C₁-C₁₂ alkylgroups (such as for example —CF), fluorinated C₁-C₁₂ aryl groups (suchas for example —C₆F₅) or fluorinated C₁-C₁₂ alkylaryl groups (such asfor example -[3,5-(CF₃)₂(C₆H₃)]).

The compound of Formula (IVa) is preferred. In this compound, the triplebond is located on the 4-position of the cyclooctyne moiety, i.e.opposite to the fused cyclobutyl ring.

Compounds with m=1 and Z═N

The present invention also relates to compounds of the general Formula(Ia), (Ib) or (Ic) wherein in is 1 and Z is N. These compounds have thegeneral Formula (Va), (Vb) or (Vc):

wherein Y is C(O) or C(R⁵)₂, and n, p, L, Q, R¹, R² and R⁵ are asdefined above.

In the compounds according to Formula (Va), (Vb) or (Vc) as shown here,the N-atom of the 4-membered ring is positioned on the 9-position of thebicyclic system. However, the present invention also relates to theregioisomers of Formula (Vb) or (Vc) wherein the N-atom of the4-membered ring is positioned on the 10-position of the bicyclic system.In addition, the two R¹-substituents may be positioned cis or trans withrespect to each other.

In one embodiment, p is 0, i.e. Q is bonded directly to the 4-memberedring. In another embodiment, p is 1. In yet another embodiment, p is 1and L is CH₂.

Also in compounds of the Formula (Va), (Vb) or (Vc), both when p is 0and when p is 1, Q is preferably selected from the group consisting of—CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)_(3,) —C(X)N(R⁶)₂,—C(R⁶)₂XR⁶, —C(X)R⁶, —C(X)XR⁶, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂,—N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, wherein X and R⁶ areas defined above. More preferably, X is oxygen. Most preferably, Q isselected from the group consisting of —OR⁶, —N(R⁶)₂, —⁺N(R⁶)₃,—C(O)N(R⁶)₂, —C(O)OR⁶, —OC(O)R⁶, —OC(O)OR⁶, —OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶,—N(R⁶)C(O)OR⁶ and —N(R⁶)C(O)N(R⁶)₂. Furthermore, the functional group Qmay optionally be masked or protected. The R⁶ groups may be selectedindependently from each other, which means that the two R⁶ groupspresent in, for example, a —N(R⁶)₂ substituent may be different fromeach other.

If Y is C(R⁵)₂, R⁵ is preferably hydrogen. Both when Y is C(R⁵)₂, andwhen Y is C(O), it is preferred that R¹ is hydrogen.

In another preferred embodiment, n is 0. In yet another preferredembodiment, R² is an electron-withdrawing group, i.e. a group with apositive value for the Hammett substituent constant σ. Suitableelectron-withdrawing groups are known to a person skilled in the art,and include for example halogen (in particular F), —OR⁶, —NO₂, —CN,—S(O)₂R⁶, substituted C₁-C₁₂ alkyl groups, substituted C₁-C₁₂ arylgroups or substituted C₁-C₁₂ alkylaryl groups, wherein the substituentsare electron-withdrawing groups. Preferably, the substituted alkylgroups, aryl groups and alkylaryl groups are fluorinated C₁-C₁₂ alkylgroups (such as for example —CF₃), fluorinated C₁-C₁₂ aryl groups (suchas for example —C₆F₅) or fluorinated C₁-C₁₂ alkylaryl groups (such asfor example -[3,5-(CF₃)₂(C₆H₃)]).

As explained above, the compound of Formula (Va) wherein the triple bondis located on the 4-position of the cyclooctyne moiety, i.e. opposite tothe fused cyclobutyl ring, is preferred.

Compounds with m=1 and Y and Z Form a Cyclic Alkyl or (Hetero)Aryl Group

The invention also relates to compounds of general Formula (Ia), (Ib) or(Ic) wherein, when m=1, Y and Z together with the bond connecting Y andZ, form a cyclic alkyl group or a (hetero)aryl group, wherein the cyclicalkyl group and the (hetero)aryl group are optionally substituted,provided that when Y and Z form a (hetero)aryl group, then the group-(L)_(p)-Q is a substituent on the (hetero)aryl group.

It is preferred that the cyclic group is an aryl group, in particular abenzannulated aryl group as in Formula (VIa), (VIb) and (VIc):

wherein p, n, R¹, R², L and Q are as defined above.

The -(L)_(p)-Q substituent may be located on either the ortho or themeta position of the benzannulated aryl group, and also in compounds ofthe Formula (VIa), (VIb) and (VIc) the R¹-substituents may be positionedcis or trans relative to each other.

In one embodiment, p is 0, i.e. Q is bonded directly to the aryl group.In another embodiment, p is 1. In yet another embodiment, p is 1 and Lis CH₂.

Q is preferably selected, both for p is 0 as for p is 1, from the groupconsisting of —CN, —N₃, —NCX, —XCN, —XR⁶, —N(R⁶)₂, —⁺N(R⁶)₃,—C(X)N(R⁶)₂, —C(R⁶)₂XR⁶, —C(X)R⁶, —C(X)XR⁶, —XC(X)R⁶, —XC(X)XR⁶,—XC(X)N(R⁶)₂, —N(R⁶)C(X)R⁶, —N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, whereinX and R⁶ are as defined above. More preferably, X is oxygen. Mostpreferably, Q is selected from the group consisting of —OR⁶, —N(R⁶)₂,—⁺N(R⁶)₃, —C(O)N(R⁶)₂, —C(O)OR⁶, —OC(O)R⁶, —OC(O)OR⁶, —OC(O)N(R⁶)₂,—N(R⁶)C(O)R⁶, —N(R⁶)C(O)OR⁶ and —N(R⁶)C(O)N(R⁶)₂. Furthermore, thefunctional group Q may optionally be masked or protected. The R⁶ groupsmay be selected independently from each other, which means that the twoR⁶ groups present in, for example, a —N(R⁶)₂ substituent may bedifferent from each other.

In a preferred embodiment, R¹ is hydrogen. In another preferredembodiment, n is 0. In yet another preferred embodiment, R² is anelectron-withdrawing group, i.e. a group with a positive value for theHammett substituent constant σ. Suitable electron-withdrawing groups areknown to a person skilled in the art, and include for example halogen(in particular F), —OR⁶, —NO₂, —CN, —S(O)₂R⁶, substituted C₁-C₁₂ alkylgroups, substituted C₁-C₁₂ aryl groups or substituted C₁-C₁₂ alkylarylgroups, wherein the substituents are electron-withdrawing groups.Preferably, the substituted alkyl groups, aryl groups and alkylarylgroups are fluorinated C₁-C₁₂ alkyl groups (such as for example —CF₃),fluorinated C₁-C₁₂ aryl groups (such as for example —C₆F₅) orfluorinated C₁-C₁₂ alkylaryl groups (such as for example-[3,5-(CF₃)₂(C₆H₃)]).

As explained above, the compound of Formula (VIa) wherein the triplebond is located on the 4-position of the cyclooctyne moiety ispreferred.

Conjugates

The fused cyclooctyne compounds according to the present invention arevery suitable for use in metal-free click reactions, and consequentlythese compounds are versatile tools in applications such as for examplebioorthogonal labeling, imaging and/or modification, including surfacemodification, of a large range of target molecules. It is an aspect ofthe present invention to provide a conjugate wherein a compound of theFormula (Ia), (Ib) and/or (Ic) according to the invention is conjugatedto a label via a functional group Q.

The term “label” refers to any identifying tag that may be conjugated toa compound of the Formula (Ia), (Ib) and (Ic) according to theinvention. A wide variety of labels are known in the art, for a widevariety of different applications. Depending on the specificapplication, a suitable label for that specific application may beselected. Suitable labels for specific applications are known to theperson skilled in the art, and include, but are not limited to, allkinds of fluorophores, biotin, polyethylene glycol (PEG) chains,polypropylene glycol (PPG) chains, mixed polyethylene/polypropyleneglycol chains, radioactive isotopes, steroids, pharmaceutical compounds,lipids, peptides, glycans (including oligo- and polysaccharides),nucleotides (including oligo- and polynucleotides) and peptide tags.Examples of suitable fluorophores are for example all kinds of AlexaFluor (e.g. Alexa Fluor 555), cyanine dyes (e.g. Cy3 or Cy5), coumarinderivatives, fluorescein, rhodamine, allophycocyanin, chromomycin, andso on. Examples of suitable peptide tags include FLAG or HIS tags. Anexample of a suitable glycan is concanavalin. Preferably, the label isselected from the group comprising fluorophores, biotin, polyethyleneglycol chains, polypropylene glycol chains, mixedpolyethylene/polypropylene glycol chains, radioactive isotopes,steroids, pharmaceutical compounds, lipids, peptides, glycans,nucleotides and peptide tags.

Functional group Q may be connected to the label directly, or indirectlyvia a linker or linking unit. Linking units are well know in the art,and have the general structure Q-S-Q, wherein Q is as defined above, andS is selected from the group consisting of linear or branched C₁-C₂₀₀alkylene groups, C₂-C₂₀₀ alkenylene groups. C₂-C₂₀₀ alkynylene groups,C₃-C₇₀₀ cycloalkylene groups. C₅-C₂₀₀ cycloalkenylene groups, C₈-C₂₀₀cycloalkynylene groups, C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀arylalkylene groups, C₈-C₂₀₀ arylalkenylene groups, C₉-C₂₀₀arylalkynylene groups. Optionally the alkylene groups, alkenylenegroups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups may be substituted, andoptionally said groups may be interrupted by one or more heteroatoms,preferably 1 to 100 heteroatoms, said heteroatoms preferably beingselected from the group consisting of O, S and NR⁶, wherein R⁶ isdefined as above. Most preferably, the heteroatom is O.

Examples of suitable linking units include, but are not limited to,(poly)ethylene glycol diamines (such as for example1,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethyleneglycol chains), polyethylene glycol or polyethylene oxide chains,polypropylene glycol or polypropylene oxide chains, 1,x-diaminoalkaneswherein x is the number of carbon atoms in the alkane, and the like.Another class of suitable linkers comprises cleavable linkers. Cleavablelinkers are well known in the art.

In a preferred embodiment, the invention relates to a conjugate, whereina compound of the Formula (IIa), (IIb) and/or (IIc) is conjugated to alabel via a functional group Q. In a further preferred embodiment, theconjugated compound of the Formula (IIa), (IIb) and/or (IIc) is acompound wherein p is 1 and L is CH₂. R¹ and/or R³ are preferably H. Inanother embodiment, n is 0 (Q, n, p, L, R¹ and R³ as defined above).Most preferably, p is 1, L is CH₂, R¹ is H, R³ is H and n is 0.

In another embodiment, the invention relates to a conjugate, wherein acompound of the Formula (IIIa), (IIIb) and/or (IIIc) is conjugated to alabel via a functional group Q. In a further embodiment, the conjugatedcompound of the Formula (IIIa), (IIIb) and/or (IIIc) is a compoundwherein p is 1 and L is CH₂. In yet another embodiment, p is 0, thusfunctional group Q is bonded directly to the N-atom. R¹ is preferably H.In another embodiment, n is 0 (Q, n, p, F, R¹ and R³ as defined above).In a most preferred embodiment, p is 1, L is CH₂, R¹ is H and n is 0. Inan alternative most preferred embodiment, p is 0, R¹ is H and n is 0.

In yet another embodiment, the invention relates to a conjugate, whereina compound of the Formula (IVa), (IVb) and/or (IVc) is conjugated to alabel via a functional group Q. In a preferred embodiment, theconjugated compound of the Formula (IVa), (IVb) and/or (IVc) is acompound wherein p is 1 and L is CH₂. R¹ and/or R³ are preferably H, andY is preferably C(O) or O. In another embodiment, n is 0 (Q, n, p, L, R¹and R³ as defined above). In a most preferred embodiment Y is O or C(O),p is 1, L is R¹ is H, R³ is H and n is 0. In another most preferredembodiment Y is O or C(O), p is 0, R¹ is R³ is H and n is 0.

In addition, the invention relates to a conjugate, wherein a compound ofthe Formula (Va), (Vb) and/or (Vc) is conjugated to a label via afunctional group Q. In a preferred embodiment, the conjugated compoundof the Formula (Va), (Vb) and/or (Vc) is a compound wherein p is 1 and Lis CH₂. R¹ preferably is H. Y is preferably C(O). In another embodiment,n is 0 (Q, n, p, F, Y, R¹ and R³ as defined above). In a most preferredembodiment Y is C(O), p is 1, L is CH₂, R¹ is H and n is 0. In anothermost preferred embodiment Y is C(O), p is 0, R¹ is H and n is 0.

The invention also relates to a conjugate, wherein a compound of theFormula (VIa), (VIb) and/or (VIc) is conjugated to a label via afunctional group Q. In a preferred embodiment, R¹ is H. In anotherpreferred embodiment, p is 0, i.e. Q is bonded directly to the arylgroup.

The present invention also relates to the use of a conjugate accordingto the invention for bioorthogonal labeling, imaging or modification,such as for example surface modification, of a target molecule.

Synthesis Method

It is a further aspect of the present invention to provide a synthesismethod for compounds of the formula (Ia), (Ib) and (Ic). The inventionthus relates to a method for preparing a compound of the general Formula(Ia), (Ib) or (Ic), the method comprising the steps of:

-   -   (a) Introduction of a fused 3- or 4-membered ring to a        cyclooctadiene of the Formula (VIIa), (VIIb) or (VIIc):

-   -   -   wherein:        -   n=0 to 8;        -   R¹ is independently selected from the group consisting of            hydrogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups,            C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl            groups; and        -   R² is independently selected from the group consisting of            halogen, —OR⁶, —NO₂, —CN, —S(O)₂R⁶, C₁-C₁₂ alkyl groups,            C₁-C₁₂ aryl groups, C₁-C₁₂ alkylaryl groups and C₁-C₁₂            arylalkyl groups, wherein the alkyl groups, aryl groups,            alkylaryl groups and arylalkyl groups are optionally            substituted, and wherein R⁶ is independently selected from            the group consisting of hydrogen, halogen, C₁-C₂₄ alkyl            groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl            groups and C₇-C₂₄ (hetero)arylalkyl groups.

    -   to form a bicyclic cyclooctene compound,

    -   (b) Bromination of the obtained bicyclic cyclooctene compound to        form a bicyclic cyclooctane compound, and

    -   (c) Dehydrobromination of the obtained bicyclic cyclooctane        compound to form a compound of the general Formula (Ia), (Ib) or        (Ic).

The cyclooctadiene of the formula (VIIa), (VIIb) or (VIIc) in step (a)may be a cis,cis- or a cis,trans-cyclooctadiene. In one embodiment, thecyclooctadiene is a cic,cis-cyclooctadiene. In a second embodiment, thecyclooctadiene is a cis,trans-cyclooctadiene. In other words, the doublebond comprising the R¹-substituents may have the E- or theZ-configuration.

In a preferred embodiment R¹ is H. In another preferred embodiment, n is0. In yet another preferred embodiment R¹ is H and n is 0.

Functional group Q may be introduced at any point in the synthesismethod. For example, the functional group may be introduced during step(a), the formation of the bicyclic cyclooctene compound. The functionalgroup may also be introduced in an additional step, for example beforeor after the bromination step (b) or after the dehydrobromination step(c). As will be clear to a person skilled in the art, the strategyemployed for the introduction of the functional group depends entirelyon the nature of the specific functional group that needs to beintroduced.

Obviously, the method of choice for step (a), the introduction of afused 3- or 4-membered ring onto a cyclooctadiene of the formula (VIIa),(VIIb) or (VIIc), depends on the type of 3- or 4-membered ring that isintroduced. A 3-membered ring comprising a N-atom may for example befused to the cyclooctadiene via reaction with a nitrene-comprisingcompound to form compounds of the Formula (IIIa), (IIIb) or (IIIc). Theintroduction of a 4-membered ring to the cyclooctadiene to formcompounds of the Formula (IVa), (IVb) or (IVc) wherein Y is C(O) may forexample be accomplished by reaction of the cyclooctadiene with aketene-comprising compound. The introduction of a 4-membered ringcomprising a N-atom to the cyclooctadiene to form compounds of theFormula (Va), (Vb) or (Vc) wherein Y is C(O), i.e. β-lactam comprisingcompounds, may be accomplished via reaction of the cyclooctadiene withan (activated) isocyanate. Subsequent hydrogenation results in compoundsof the Formula (Va), (Vb) or (Vc) wherein Y is CH₂. Compounds of theFormula (VIa), (VIb) or (VIc) may for example be prepared via reactionof the cyclooctadiene with a (substituted) benzyne compound.

Step (b), bromination of a cyclooctane compound, and step (c),dehydrobromination of a cyclooctane compound, are considered standardorganic transformations that are well known to a person skilled in theart.

The present inventors found that for example compounds of the Formula(IIa), (IIb) or (IIc) can be easily and rapidly prepared in good yieldfrom readily available starting materials. The present inventiontherefore also relates to a method for preparing a compound of theFormula (IIa), (IIb) or (IIc), the method comprising the steps of:

-   -   (a) Cyclopropanation of a cyclooctadiene of the Formula (VIIa),        (VIIb) or (VIIc):

-   -   wherein n, R¹ and R² are as defined above to form a bicyclic        cyclooctene compound,    -   (b) Bromination of the obtained bicyclic cyclooctene compound to        form a bicyclic cyclooctane compound, and    -   (c) Dehydrobromination of the obtained bicyclic cyclooctane        compound to form a compound of the Formula (IIa), (IIb) or        (IIc).

The cyclooctadiene of the Formula (VIIa), (VIIb) or (VIIc) in step (a)may be a cis,cis- or a cis,trans-cyclooctadiene. In one embodiment, thecyclooctadiene is a cic,cis-cyclooctadiene. In another embodiment, thecyclooctadiene is a cis,trans-cyclooctadiene. In other words, the doublebond comprising the R¹-substituents may have the E- or theZ-configuration.

In a preferred embodiment R¹ is H. In another preferred embodiment, n is0. In yet another preferred embodiment R¹ is H and n is 0.

Also in this case, functional group Q may be introduced in an additionalstep, for example before or following bromination step (b), or followingdehydrobromination step (c). Step (a), the cyclopropanation of acyclooctadiene may for example be achieved by reaction of a carbene,carbenoid or a carbene precursor with the cyclooctadiene, optionally inthe presence of a catalyst. Step (b) and (c) are standard organictransformations that are well known to the person skilled in the art.

As an example, a compound of the Formula endo-(IIa), wherein p is 1, Lis CH₂, Q is OH, n is 0, and R¹ and R³ are H(endo-9-(hydroxymethyl)bicyclo[6.1.0]non-4-yne), may be synthesised ingood yield in only 4 steps in a very short time (only 1-2 days). Yetanother advantage of the synthesis method according to the presentinvention is that there only is a limited need for chromatographicpurification, thus reducing the total time needed for the totalsynthesis.

Modification of Target Molecules

The conjugates according to the present invention are successfullyapplied in bioorthogonal labeling, imaging or modification, includingsurface modification, of target molecules such as for example proteins,lipids and glycans. The present invention therefore also relates to amethod for the modification of a target molecule, wherein a conjugateaccording to the present invention is reacted with a compound comprisinga 1,3-dipole or a 1,3-(hetero)diene. As an example, the strain-promotedcycloaddition of a cycloalkyne with an azide (SPAAC) or with a nitrone(SPANC) was depicted in Scheme 1. The reaction of a cyclooctyne with a1,3-(hetero)diene is known as a (hetero) Diels-Alder reaction. Thesereactions are also referred to as metal-free click reactions.

1,3-Dipolar compounds are well known in the art (cf. for example F. A.Carey and R. J. Sundberg, Advanced Organic Chemistry, Part A: Structureand Mechanisms, 3^(rd) Ed., 1990, p. 635-637), and include nitrileoxides, azides, diazomethane, nitrones, nitrilamines, etc. Preferably,the compound comprising a 1,3-dipole is an azide-comprising compound, anitrone-comprising compound or a nitrile oxide-comprising compound.

(Hetero) Diels-Alder reactions and 1,3-(hetero)dienes are also wellknown in the state of the art. Examples of 1,3-dienes include, amongstothers, 1,3-butadiene, 1,3-cyclopentadiene, 1,3-cyclohexadiene, furan,pyrrole, and their substituted varieties. Examples of 1,3-heterodienesinclude amongst others 1-oxa-1,3-butadiene, 1-aza-1,3-butadiene,2-aza-1,3-butadiene, 3-aza-1,3-butadiene, and their substitutedvarieties.

A large variety of target molecules, i.e. compounds comprising a1,3-dipole or a 1,3-(hetero)diene, may be modified by the methodaccording to the invention. Suitable target molecules are well known inthe art and include, but are not limited to, biomolecules such as forexample proteins, peptides, glycans, lipids, nucleic acids, enzymes,hormones, and the like. In principle, any compound comprising a1,3-dipole or a 1,3-(hetero)diene may be suitable as a target molecule.

Applications of the method for the modification of target moleculesaccording to the present invention include, but are by no means limitedto, diagnostic and therapeutic applications, cell labeling of livingcells, for example MV3 cells, fibroblasts, Jurkat, CHO or HEK cells,modification of biopolymers (proteins, lipids, nucleic acids, glycans),enrichment of proteins and glycans for mass spectrometric analysis,tuning of polymer properties, surface modifications etc.

In one embodiment, the reaction of the conjugate is performed in vitro.In a preferred embodiment, the reaction is performed in vivo, i.e. underphysiological conditions.

The conjugates according to the present invention that are applied inthe modification of a target molecule are described above in greatdetail. One of the large advantages of these conjugates is that they maybe applied both in vivo and in vitro. In addition, the here describedconjugates suffer less from undesired aspecific lipophilic interactions,and show good reaction kinetics in metal-free click reactions. Anotheradvantage is that the here described conjugates are easily synthesizedand amenable to simple and straightforward modification of various partsof the conjugate. This makes it possible to “fine tune” a conjugate fora specific application, and optimise reaction kinetics for thisapplication.

In a preferred embodiment, a compound of the Formula (IIa), (IIb) and/or(IIc), or a conjugate thereof as described above, is reacted with acompound comprising a 1,3-dipole or a 1,3-(hetero)diene. For example thecycloaddition of (IIa) with benzyl azide in aqueous conditions proceedsrapidly and cleanly to the corresponding triazole adducts with excellentreaction kinetics (k=0.09-0.28 M⁻¹ s⁻¹, depending on the solvent).Reaction kinetics for the cycloaddition of (IIa) withC-benzylamide-N-methylnitrone are even higher (k=1.25 M⁻¹ s⁻¹).Surprisingly, the reaction kinetics are similar for both exo-(IIa) andendo-(IIa), indicating that the stereochemistry at the C-9 position ofcompound (IIa) has little influence on reactivity. Consequently, amixture of the exo- and endo-compounds according to the invention may beapplied in some applications, avoiding the need for separation of theexo- and endo-compounds and even further simplifying the synthesis ofthe compounds according to the invention.

Additional applications of the method according to the present inventioninclude for example the ligation of a fluorophore conjugate wherein(IIa) is conjugated with Alexa Fluor 555 to a viral plasmid proteincontaining a single azide, and the detection of cell surface glycans bymeans of the chemical reporter strategy.

Pharmaceutical Composition

Finally, the invention relates to a composition comprising a conjugateaccording to the invention, further comprising a pharmaceuticallyacceptable carrier. A wide variety of suitable pharmaceuticallyacceptable carriers are known in the art (cf. for example R. C. Rowe, P.J. Sheskey and P. J. Weller (Eds.), Handbook of PharmaceuticalExcipients, 4^(th) Ed. 2003).

EXAMPLES Example 1 Synthesis of exo- and endo-IIa.2

In the first step, ethyl diazoacetate was slowly added to a solution indichloromethane of a large excess (8 equiv.) of 1,5-cyclooctadiene inthe presence of rhodium acetate, leading to the diastereomeric compoundsexo-IIa.1 and endo-IIa.1 in a ratio of 2:1 (combined yield of 82%).After straightforward separation by silica gel, first the endo-isomer ofIIa.1 was converted into alcohol IIa.2 following a straightforwardthree-step procedure of reduction, bromination and elimination. Thus,ester reduction of endo-IIa.1 with LiAlH₄ (30 min) gave a crudeintermediate alcohol (30 min) that was pure enough for brominationwithout intermediate purification (30 min). It must be noted thattemporary protection of the alcohol, as in the synthesis of DIBO, is notrequired. Finally, the resulting dibromide was subjected to excess KOtBuin THF (0° C.→reflux, 2 h), affording the desired9-(hydroxymethyl)bicyclo[6.1.0]non-4-yne endo-IIa.2 in 61% isolatedyield for the three steps from endo-IIa.1, A similar sequence of events,that can be executed in a single day with only one chromatographicpurification, afforded the diastereomeric exo-isomer of IIa.2 in 53%.

Example 2 Reaction Kinetics

The reaction kinetics for cycloaddition with the prototypical azidebenzyl azide were investigated. Thus, compound endo-IIa.2 was dissolvedin a 3:1 mixture of CD₃CN and D₂O and mixed with benzyl azide at a finalconcentration of 18 mM. Chemical conversion was followed with proton NMRby integration of diagnostic peaks and indicated that endo-IIa.2 reactedrapidly and cleanly to the expected triazole adducts. Calculation ofsecond-order reaction kinetics showed a rate constant of 0.12 s⁻¹. Asimilar value of 0.10 M⁻¹ s⁻¹ was determined for exo-IIa.2, indicatingthat the stereochemistry at the C-9 position has little influence onreactivity, with only a slightly higher rate for the endo-configuredcompounds. A significant solvent effect Was noticed because the samecycloaddition reaction in a 1:2 mixture of CD₃CN and D₂O gave rateconstants of 0.28 and 0.25 M⁻¹ s⁻¹ for endo-IIa.2 and exo-IIa.2,respectively.

Example 3 Bicyclo[6.1.0]non-4-yne (BCN) Conjugates

BCN Conjugated to Biotin (IIa.4)

The alcohol moiety of endo-IIa.1 was converted into a p-nitrophenyl(pNP) carbonate as in IIa.3. Subsequent reaction with biotin-(POE)₃-NH₂led to BCN-biotin conjugate IIa.4 (69% yield).

BCN Conjugated to Alexa Fluor 555 (IIa6)

Alternatively, compound IIa.3 was reacted with1,8-diamino-3,6-dioxaoctane to give amino-terminated BCN-probe IIa.5,that was converted into the fluorophore conjugate IIa.6 upon reactionwith commercial Alexa Fluor 555 hydroxysuccinimide ester.

Alternatively, compound IIa.3 was reacted with 1,5-diaminopentane togive a more lipophilic amino-terminated BCN-probe.

Example 4 Reaction of IIa.6 with Azide-Labeled Capsid Protein (SPAACLabeling)

The capsid protein was prepared to contain only a single azidefunctionality per molecule, which—after assembly of the capsid—islocated on the inside surface of the capsid. The protein is dissolved inbuffer (50 mM phosphate buffer pH 7.5; 1M NaCl) at a concentration of 1mg/mL, Alexa Fluor 555-BCN conjugate IIa.6 was dissolved in water and 4equivalents of the conjugate were added to the protein solution. Thereaction mixture was incubated for 3 hours at RT before analysis.

The reaction product was analyzed on a 12% SDS-PAGE gel, of which afluorescence image was recorded before staining with Coomassie-blue. InFIG. 1 the SDS-Page analyses of the reaction product (left) and of theblank reaction (right) are shown. The top part of FIG. 1 shows theCoomassie-blue staining, the bottom part shows the fluorescence imagebefore staining with Coomassie-blue. These results clearly indicate thatthe Alexa Fluor 555-BCN conjugate was incorporated into the capsidprotein.

For mass analysis, the crude reaction mixture was dialyzed to 0.1% TFAin milliQ before analyzing it by electron-spray ionizationtime-of-flight (ESI-TOF) on a JEOL AccuTOF. FIG. 2 shows the ESI-TOFmass spectrum of capsid protein reacted with IIa.6. Peak B correspondsto the mass of unreacted capsid protein, while peak A corresponds to thereaction product. The mass difference between the peaks is 1135.1(expected 1135), indicating that the single azide in the capsid proteinhas effectively reacted with the BCN-Alexa Fluor555 conjugate which hasa mass of 1135.

To test the self-assembly properties of the functionalized capsidprotein, the crude reaction mixture was dialyzed to a buffer at pH 5.0(50 mM NaOAc; 1 M NaCl; 10 mM CaCl₂), which should induce the formationof 28 nm sized spherical particles (B. J. M. Verduin, FEBS Lett, 1974,45, 50-54, incorporated by reference). After assembly of the capsids bydialysis, the reaction mixture was analyzed by FPLC. The size exclusionanalysis on a Superpose 6 column is shown in FIG. 3. The absorption peakat 280 nm at an elution volume of 1.2 ml indicates the formation of 28nm-sized capsids, and the overlapping absorbance at 555 nm shows thatthe Alexa dye, and thus IIa.6, is present in the capsids.

Example 5 Reaction of IIa.2 with Nitrone-Labeled FRATtide Protein (SPANCLabeling)

FRATtide (15.6 μg, 3.4 nmol, 34 μM) was dissolved in 0.1 M NH₄OAc bufferpH 6.9 (100 μL) and NaIO₄ (1.1 μg, 5.5 nmol, 48 μM) was added. Thereaction was allowed to take place at room temperature for 40 min andp-methoxybenzenethiol (9.2 μg, 66.0 nmol, 565 μM) was added. The mixturewas shaken for 2 h at 25° C. and p-anisidine (13.5 μg, 109.3 μmol, 845μM), N-methylhydroxylamine hydrochloride (18.2 μg, 218.6 nmol, 1.5 mM),and BCN—OH II.a2 (41.1 μg, 273.3 nmol, 1.8 mM) were added. Finally, thereaction mixture was shaken at 25° C. for 24 h to give the desiredconjugate.

Progress of the reaction was monitored by mass spectrometry (Accu-TOF).The molecular ion peak at 4533.8 Da, corresponding to the FRATtideprotein, disappeared upon treatment with sodium periodate, whereas amolecular ion peak at 4534.8, corresponding to the methyl hemiacetal,appeared. Upon treatment with N-methylhydroxylamine and BCN—OH, amolecular ion peak appeared at 4681.4 Da, corresponding to FRATtideprotein nitrone derivative that has reacted with BCN—OH IIa.2, as themain peak (after deconvolution).

Example 6 Cell-Surface Labeling

The usefulness of biotin-conjugate IIa.4 for bioorthogonal labelingpurposes was investigated by detection of cell surface glycans by meansof the chemical reporter strategy. N-Azidoacetylmannosamine (ManNAz) wasmetabolically incorporated in MV3 cells, and detected by FACS andconfocal microscopy.

Cell Culture Procedure

Invasive and metastatic human melanoma cells (MV3) were maintained inculture medium RPMI 1640, containing 10% fetal calf serum,penicillin/streptomycin (each 50 U/ml) in a 5% CO₂ water-saturatedatmosphere.

Cell Surface Azide Labeling

MV3 cells were cultured for 6 days in the absence or presence ofAc₄ManNAz (50 μM). Medium and compound change was performed after 3days. Cell adhesion and morphology was analyzed with a bright fieldmicroscope prior to the advanced studies. For live-cell labeling, cellswere detached by EDTA (1 mM), washed and centrifuged three times (PBS,300×G rpm, 5 min, 4° C.), resuspended in PBS and incubated in BCN-biotin(IIa.4) (60 μM), DIBO-biotin (60 μM) or buffer (1 h, 20° C.), washedthree times (PBS, 300×G, 2 min, 4° C.), resuspended in ice-cold PBScontaining AlexaFluor488-conjugated streptavidin (5 μg/ml, Invitrogenfinal volume 200 μl). After incubation (30 min, 4° C.), cells werewashed three times, resuspended in PBS (200 μl, 4° C.) for furtherbiological analysis.

Flow Cytometry

Flow cytometry was performed on a BD Biosciences FACS-Calibur flowcytometer using the 488 nm argon laser and data were analyzed with FCSExpress version 3 research edition (De Novo Software, Los Angeles,Calif.). Per sample, 2×10⁴ morphologically intact cells were analyzed inthe presence of propidium iodide (2.5 μg/ml).

In FIG. 4, the cell surface fluorescence on intact MV3 cells aftermetabolic incorporation of Ac₄ManNAz, labeling with DIBO- or BCN-biotinand detection with AlexaFluor488-conjugated streptavidin is shown.Analysis by flow cytometry indicates a more than 100-fold increase influorescence intensity for labeling with BCN-biotin or DIBO-biotin,followed by detection with AlexaFluor488-conjugated streptavidin, withneglible background levels of fluorescence when cells are not incubatedwith Ac₄ManNAz.

FIG. 5 shows the fluorescence intensities and cell viability of MV3cells after Metabolic incorporation of Ac₄ManNAz, labeling with DIBO- orBCN-biotin, and detection with AlexaFluor488-conjugated streptavidin. InFIG. 5A the mean intensities for green fluorescence (AlexaFluor488) andstandard deviations (SD) from four independent experiments are shown, inFIG. 5B the intact cell viability after glycan labeling. Greenfluorescence (AlexaFluor488) and propidium iodide (PI) labels were used.Numbers indicate the percentage of PI-negative, viable cells. From FIG.5A, it can be concluded that both BCN and DIBO are effectively labelingcells that were prior exposed to Ac₄ManNAz, with a higher efficiency oflabeling with BCN-biotin. FIG. 5B shows that cells, whether untreated,treated with BCN or treated with DIBO, showed high viability (>98% inall cases).

Confocal Microscopy

Cell surface glycans were labeled with BCN-biotin or DIBO-biotin andnext with AlexaFluor488-conjugated streptavidin. After labeling, cellswere resuspended in normal culture medium, transferred in a 6-well plateand incubated for 30 min at 37° C. Life cell imaging of MV3 cells wasperformed on a Olympus FV1000 confocal laser scanning microscope with anargon 488 laser, excitation 488 nm, emission 520 nm and a 40×magnification at room temperature.

FIG. 6 shows representative confocal images of labeled cells, previouslycultured in absence or presence of Ac₄ManNAz (50 nM). The confocalimages clearly show that the cell surface of MV3-cells becomefluorescent upon incubation of the cells with Ac₄ManNAz followed bydetection of cell surface glycans with BCN-biotin or DIBO-biotin andthen AlexaFluor488-conjugated streptavidin, but not without incubationwith Ac₄ManNAz.

Example 7 Synthesis of IIa.7

(1R,8S,Z)-Diethyl bicyclo[6.1.0]non-4-ene-9,9-dicarboxylate (IIa.8)

To a solution of 1,5-cyclooctadiene (5.27 mL, 43.0 mmol) and Rh₂(OAc)₄(100 mg, 0.23 mmol) in CH₂Cl₂ (5 mL) was added dropwise in 3 h asolution of diethyl diazomalonate (1.0 g, 5.37 mmol) in CH₂Cl₂ (5 mL).This solution was stirred for 24 h at rt. The CH₂Cl₂ was evaporated andthe excess of cyclooctadiene was removed by filtration over a glassfilter filled with silica (eluens: heptane). The filtrate wasconcentrated in vacuo and the residue was purified by columnchromatography on silica gel (EtOAc:heptane, 1:10) to afford IIa.8 (1.03g, 72%).

¹H NMR (CDCl₃, 400 MHz): δ 5.65-5.57 (m, 2H), 4.10 (2×q, J==7.2 Hz, 4H),2.41-2.29 (m, 2H), 2.15-2.06 (m, 3H), 1.83-1.70 (m, 3H), 1.31-1.23 (2×t,J=7.2 Hz, 6H).

(1,R,8S,Z)-Bicyclo[6.1.0]non-4-ene-9,9-diyldimethanol (IIa.9)

To a suspension of LiAlH₄ (103 mg, 2.70 mmol) in Et₂O (10 mL) was addeddropwise at 0° C. a solution of IIa.8 (400 mg, 1.50 mmol) in Et₂O (10mL). Water was added carefully until the grey solid had turned intowhite. Then Na₂SO₄ (2 g) was added, the solid was filtered off andwashed thoroughly with Et₂O (100 mL). The filtrate was concentrated invacuo. The residue was purified by column chromatography on silica gel(EtOAc:heptane, 3:1) to afford IIa.9 as a white solid (190 mg, 69%).

¹H NMR (CDCl₃, 400 MHz): δ 5.66-5.58 (m, 2H), 3.88 (d, J=4.8 Hz, 2H),3.58 (d, J=4.8 Hz, 2H), 2.43-2.35 (m, 2H), 2.20-1.99 (m, 6H), 1.71-1.57(m, 2H), 0.95-0.88 (m, 2H).

(1R,8S)-4,5-Dibromobicyclo[6.1.0]nonane-9,9-diyl)dimethanol (IIa.10

The diol IIa.9 (145 mg, 0.796 mmol) was dissolved in CH₂Cl₂ (5 mL). At0° C. a solution of Br₂ (45 μL, 0.875 mmol) in CH₂Cl₂ (1 mL) was addeddropwise until the yellow color persisted. The reaction mixture wasquenched with a 10% Na₂S₂O₃-solution (5 mL), and extracted with CH₂Cl₂(2×20 mL). The organic layer was dried (Na₂SO₄) and concentrated invacuo. The residue was purified by column chromatography on silica gel(EtOAc:heptane, 5:1) afford IIa.10 (235 mg, 86%) as a white solid.

¹H NMR (CDCl₃, 400 MHz): δ 4.87-4.78 (m, 2H), 3.96-3.88 (m, 2H), 3.60(d, J=5.2 Hz, 2H), 2.75-2.63 (m, 2H), 2.32-2.22 (m, 3H), 2.20-2.13 (m,1H), 2.05-1.94 (m, 2H), 1.74-1.57 (m, 2H), 1.13-0.99 (m, 2H).

(1R,8S)-Bicyclo[6.1.0]non-4-yn-9,9-diyldimethanol (IIa.7)

To a solution of the dibromide IIa.10 (100 mg, 0.292 mmol) in THF (5 mL)was added dropwise at 0° C. a solution of KOtBu (1.29 mL, 1 M in THF,1.29 mmol). Then the solution was refluxed for 1.5 h. After cooling downto it the mixture was quenched with saturated NH₄Cl-solution (20 mL),and extracted with CH₂Cl₂ (3×20 mL). The organic layer was dried(Na₂SO₄) and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (EtOAc) to afford IIa.7 (24 mg, 46%) as awhite solid.

¹H NMR (CDCl₃, 400 MHz): δ 3.89 (bs, 2H), 3.63 (bs, 2H), 2.58 (bs, 2H),2.34-2.20 (m, 6H), 1.68-1.59 (m, 2H), 0.89-0.82 (m, 2H).

The invention claimed is:
 1. A compound of the Formula (IIa):

wherein: n is 0 to 8; p is 0 or 1; R³ is selected from the groupconsisting of [(L)_(p)-Q], hydrogen, halogen, C₁-C₂₄ alkyl groups,C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄(hetero)arylalkyl groups, the alkyl groups optionally being interruptedby one of more hetero-atoms selected from the group consisting of O, Nand S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)arylgroups and (hetero)arylalkyl groups are independently optionallysubstituted with one or more substituents independently selected fromthe group consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂ alkenyl groups,C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₁-C₁₂ alkoxy groups,C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy groups, C₃-C₁₂ cycloalkyloxygroups, halogens, amino groups, oxo groups and silyl groups, wherein thealkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, alkoxygroups, alkenyloxy groups, alkynyloxy groups and cycloalkyloxy groupsare optionally substituted, the alkyl groups, the alkoxy groups, thecycloalkyl groups and the cycloalkoxy groups being optionallyinterrupted by one of more hetero-atoms selected from the groupconsisting of O, N and S, wherein the silyl groups are represented bythe formula (R⁴)₃Si—, wherein R⁴ is independently selected from thegroup consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂ alkenyl groups, C₂-C₁₂alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₁-C₁₂ alkoxy groups, C₂-C₁₂alkenyloxy groups, C₂-C₁₂ alkynyloxy groups and C₃-C₁₂ cycloalkyloxygroups, wherein the alkyl groups, alkenyl groups, alkynyl groups,cycloalkyl groups, alkoxy groups, alkenyloxy groups, alkynyloxy groupsand cycloalkyloxy groups are optionally substituted, the alkyl groups,the alkoxy groups, the cycloalkyl groups and the cycloalkoxy groupsbeing optionally interrupted by one of more hetero-atoms selected fromthe group consisting of O, N and S; L is a linking group selected fromlinear or branched C₁-C₂₄ alkylene groups, C₂-C₂₄ alkenylene groups,C₂-C₂₄ alkynylene groups, C₃-C₂₄ cycloalkylene groups, C₅-C₂₄cycloalkenylene groups, C₈-C₂₄ cycloalkynylene groups, C₇-C₂₄alkyl(hetero)arylene groups, C₇-C₂₄ (hetero)arylalkylene groups, C₈-C₂₄(hetero)arylalkenylene groups, C₉-C₂₄ (hetero)arylalkynylene groups, thealkylene groups, alkenylene groups, alkynylene groups, cycloalkylenegroups, cycloalkenylene groups, cycloalkynylene groups,alkyl(hetero)arylene groups, (hetero)arylalkylene groups,(hetero)arylalkenylene groups and (hetero)arylalkynylene groupsoptionally being substituted with one or more substituents independentlyselected from the group consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₅-C₁₂cycloalkenyl groups, C₈-C₁₂ cycloalkynyl groups, C₁-C₁₂ alkoxy groups,C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy groups, C₃-C₁₂ cycloalkyloxygroups, halogens, amino groups, oxo and silyl groups, wherein the silylgroups can be represented by the formula (R⁴)₃Si—, wherein R⁴ is definedas above; Q is a functional group selected from the group consisting ofhalogen, C₁-C₂₄ alkyl, C₆-C₂₄ (hetero)aryl, C₇-C₂₄ alkyl(hetero)aryl,C₇-C₂₄ (hetero)arylalkyl, —CH═C(R⁶)₂, —C≡CR⁶, —[C(R⁶)₂C(R⁶)₂O]_(q)—R⁶,wherein q is in the range of 1 to 200, —CN, —N₃, —NCX, —XCN, —XR⁶,—N(R⁶)₂, —⁺N(R⁶)₃, —C(X)N(R⁶)₂, —C(R⁶)₂XR⁶, —C(X)R⁶, —C(X)XR⁶, —S(O)R⁶,—S(O)₂R⁶, —S(O)OR⁶, —S(O)₂OR⁶, —S(O)N(R⁶)₂, —S(O)₂N(R⁶)₂, —OS(O)R⁶,—OS(O)₂R⁶, —OS(O)OR⁶, —OS(O)₂OR⁶, —P(O)(R⁶)(OR⁶), —P(O)(OR⁶)₂,—OP(O)(OR⁶)₂, —Si(R⁶)₃, —XC(X)R⁶, —XC(X)XR⁶, —XC(X)N(R⁶)₂, —N(R⁶)C(X)R⁶,—N(R⁶)C(X)XR⁶ and —N(R⁶)C(X)N(R⁶)₂, wherein X is oxygen or sulphur andwherein R⁶ is independently selected from the group consisting ofhydrogen, halogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups,C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl groups; R¹is independently selected from the group consisting of hydrogen, C₁-C₂₄alkyl groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄ alkyl(hetero)arylgroups and C₇-C₂₄ (hetero)arylalkyl groups; and R² is independentlyselected from the group consisting of halogen, —OR⁶, —NO₂, —CN,—S(O)₂R⁶, C₁-C₁₂ alkyl groups, C₁-C₁₂ aryl groups, C₁-C₁₂ alkylarylgroups and C₁-C₁₂ arylalkyl groups, wherein R⁶ is as defined above, andwherein the alkyl groups, aryl groups, alkylaryl groups and arylalkylgroups are optionally substituted.
 2. The compound according to claim 1,wherein n is
 0. 3. The compound according to claim 1, wherein p is 1 andL is CH₂.
 4. The compound according to claim 1, wherein Q is selectedfrom the group consisting of —OR⁶, —N(R⁶)₂, —⁺N(R⁶)₃, —C(O)N(R⁶)₂,—C(O)OR⁶, —OC(O)R⁶, —OC(O)OR⁶, —OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶, —N(R⁶)C(O)OR⁶and —N(R⁶)C(O)N(R⁶)₂, wherein R⁶ is as defined in claim
 1. 5. Thecompound according to claim 1, wherein Q is —OH.
 6. The compoundaccording to claim 1, wherein R₁ is hydrogen.
 7. The compound accordingto claim 1, wherein R³ is hydrogen or [(L)_(p)-Q].
 8. The compoundaccording to claim 1, wherein p is 1, L is CH₂, Q is —OH, R¹ is hydrogenand R³ is hydrogen or [(L)_(p)-Q].
 9. The compound according to claim 2,wherein p is 1, L is CH₂, Q is —OH, R¹ is hydrogen, and R³ is hydrogenor [(L)_(p)-Q].
 10. A conjugate comprising a compound according to claim1 conjugated to a label via a functional group Q, wherein the label isselected from the group comprising fluorophores, biotin, polyethyleneglycol chains, polypropylene glycol chains, mixedpolyethylene/polypropylene glycol chains, radioactive isotopes,steroids, pharmaceutical compounds, lipids, peptides, glycans,nucleotides and peptide tags.
 11. The conjugate according to claim 10,wherein functional group Q is connected to the label via a linking unit.12. The conjugate according to claim 11, wherein the linking unit isselected from the group consisting of linear or branched C₁-C₂₀₀alkylene groups, C₂-C₂₀₀ alkenylene groups, C₂-C₂₀₀ alkynylene groups,C₃-C₂₀₀ cycloalkylene groups, C₅-C₂₀₀ cycloalkenylene groups, C₈-C₂₀₀cycloalkynylene groups, C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀arylalkylene groups, C₈-C₂₀₀ arylalkenylene groups, C₉-C₂₀₀arylalkynylene groups, wherein optionally the alkylene groups,alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups aresubstituted, and wherein optionally said groups are interrupted by oneor more heteroatoms, said heteroatoms being selected from the groupconsisting of O, S and NR⁶, wherein R⁶ is defined as in claim
 1. 13. Theconjugate according to claim 10, wherein, in the compound according toclaim 1, p is 1, L is CH₂, R¹ is H and R³ is H.
 14. A method forpreparing a compound according to claim 1, the method comprising: (a)Cyclopropanation of a cyclooctadiene of the Formula (VIIa):

wherein: n=0 to 8; R¹ is independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups,C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl groups; andR² is independently selected from the group consisting of halogen, —OR⁶,—NO₂, —CN, —S(O)₂R⁶, C₁-C₁₂ alkyl groups, C₁-C₁₂ aryl groups, C₁-C₁₂alkylaryl groups and C₁-C₁₂ arylalkyl groups, wherein the alkyl groups,aryl groups, alkylaryl groups and arylalkyl groups are optionallysubstituted, and wherein R⁶ is independently selected from the groupconsisting of hydrogen, halogen, C₁-C₂₄ alkyl groups, C₆-C₂₄(hetero)aryl groups, C₇-C₂₄ alkyl(hetero)aryl groups and C₇-C₂₄(hetero)arylalkyl groups, to form a bicyclic cyclooctene compound, (b)Bromination of the obtained bicyclic cyclooctene compound to form abicyclic cyclooctane compound, and (c) Dehydrobromination of theobtained bicyclic cyclooctane compound to form a compound according toclaim
 1. 15. A method for the modification of a target molecule,comprising reacting a conjugate according to claim 10 with a compoundcomprising a 1,3-dipole or a 1,3-(hetero)diene.
 16. The method accordingto claim 15, wherein the compound comprising a 1,3-dipole is anazide-comprising compound, a nitrone-comprising compound, or a nitrileoxide-comprising compound.
 17. The method according to claim 15, whereinthe conjugate comprises a functional group Q, wherein the functionalgroup Q is connected to the label via a linking unit.
 18. The methodaccording to claim 17, wherein the linking unit is selected from thegroup consisting of linear or branched C₁-C₂₀₀ alkylene groups, C₂-C₂₀₀alkenylene groups, C₂-C₂₀₀ alkynylene groups, C₃-C₂₀₀ cycloalkylenegroups, C₅-C₂₀₀ cycloalkenylene groups, C₈-C₂₀₀ cycloalkynylene groups,C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀ arylalkylene groups, C₈-C₂₀₀arylalkenylene groups, C₉-C₂₀₀ arylalkynylene groups, wherein optionallythe alkylene groups, alkenylene groups, alkynylene groups, cycloalkylenegroups, cycloalkenylene groups, cycloalkynylene groups, alkylarylenegroups, arylalkylene groups, arylalkenylene groups and arylalkynylenegroups are substituted, and wherein optionally said groups areinterrupted by one or more heteroatoms, said heteroatoms being selectedfrom the group consisting of O, S and NR⁶, wherein R⁶ is defined as inclaim
 1. 19. A composition comprising a conjugate according to claim 10and a pharmaceutically acceptable carrier.
 20. The compound according toclaim 2, wherein p is 1 and L is CH₂.
 21. The compound according toclaim 2, wherein Q is selected from the group consisting of —OR⁶,—N(R⁶)₂, —⁺N(R⁶)₃, —C(O)N(R⁶)₂, —C(O)OR⁶, —OC(O)R⁶, —OC(O)OR⁶,—OC(O)N(R⁶)₂, —N(R⁶)C(O)R⁶, —N(R⁶)C(O)OR⁶ and —N(R⁶)C(O)N(R⁶)₂, whereinR⁶ is independently selected from the group consisting of hydrogen,halogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl groups.
 22. Thecompound according to claim 2, wherein Q is —OH.
 23. The compoundaccording to claim 2, wherein R₁ is hydrogen.
 24. The compound accordingto claim 2, wherein R³ is hydrogen or [(L)_(p)-Q].
 25. The compoundaccording to claim 23, wherein R³ is hydrogen or [(L)_(p)-Q].
 26. Thecompound according to claim 9, wherein R³ is hydrogen.
 27. The compoundaccording to claim 9, wherein R³ is [(L)_(p)-Q] and wherein p is 1, L isCH₂ and Q is OH.